The Mechanism Of Shengmaiyin Mediating Lipid Autophagy Against Diabetes Cardiomyopathy Through Alkbh5-Atg7 Pathway

Research background and purposeDiabetes cardiomyopathy(DCM)is a cardiovascular complication of diabetes,which has been paid more and more attention by clinicians.DCM is an independent primary disea se.In the early stage,it is characterized by diastolic dysfunction,which is dominated by dec reased myocardial compliance and blocked diastole filling.In the late stage,it is characteriz ed by cardiac systolic dysfunction,which will eventually develop into heart failure.At pres ent,there is a lack of targeted therapeutic drugs for DCM,and the treatment method is still mainly to reduce blood sugar,but cardiovascular benefits are small while reducing blood su gar,so a considerable number of patients have poor efficacy,and the incidence rate and mor tality of heart failure caused by DCM are still high.Shengmai Yin is derived from Zhang Yuanyuan’s "Medical Enlightenment" in the Jin Dynasty.It is composed of three herbs: ginseng,ophiopogon japonicus,and schisandra.It h as the functions of tonifying qi,restoring meridians,nourishing yin,and promoting fluid pr oduction.It is a representative formula for tonifying qi,nourishing yin,and generating meri dians.At present,Shengmai Yin has been made into various dosage forms for clinical treat ment.Although Shengmai Yin has a significant therapeutic effect on DCM and is safe to us e,the mechanism of action of Shengmai Yin is not clear.Therefore,further elucidating the s pecific pathogenesis of DCM and clarifying the target of action of Shengmai Yin are of grea t significance for searching for targeted therapeutic drugs for DCM and guiding clinical me dication.This topic uses the whole animal experiment to evaluate the protective effect of Sheng maiyin on the heart of diabetes.On the basis of clarifying that Shengmaiyin improves the h eart function of DCM,we explore the mechanism through cell experiments and verify it in animals.Finally,we verify the regulatory effect of Shengmaiyin on the lipid autophagy path way at the cellular and animal levels.MethodPart 1:Animal experiment: 45 C57BL/6 male mice(20 ± 2g)aged 8 weeks were used.10 mic e were randomly selected as the Control group.The Control group was fed with regular fee d,while the remaining 35 mice were fed with 60% high fat feed.All mice were given free d rinking water.After 1 month,except for the Control group,streptozotocin(STZ)60mg/kg was injected intraperitoneally for 5 consecutive days.After 3 days of injection,the tail vein was cut off to take blood.Before taking blood,the mice were starved on an empty stomach for 3 hours,and the blood sugar value was higher than 16.7 mmol/L for three consecutive ti mes to determine that the diabetes model was successfully established.In this experiment,32 mice were determined to have successfully established the diabetes model.32 mice were r andomly divided into DCM group(12 mice),Shengmaiyin low-dose group(LSMY)and Sh engmaiyin high-dose(HSMY)group(10 mice each).Two animals died in the DCM group during the experiment.After 16 weeks,mice underwent ultrasound testing and heart tissue s amples were taken.Ultrasound was used to detect cardiac function indicators such as LVES D,LVEDD,LVEF,and LVFS in mice,HE staining was used to detect pathological changes in left ventricular myocardium,Masson staining was used to detect the degree of left ventri cular myocardial fibrosis,qPCR was used to detect changes in ANP mRNA and BNP mRN A levels in mouse heart tissue,and ELISA was used to detect changes in LDH,CK-MB,cT N1,and TG in mouse serum.Part 2:Cell experiment: 1.Literature review was conducted to screen the dosages of palmitate(PA)and high concentration glucose(HG)in H9c2 myocardial cells induced by high sugar and high fat,and the levels of ANP mRNA and BNP mRNA in H9c2 myocardial cells induc ed by high sugar and high fat were detected by qPCR.2.The transcriptome sequencing of H9c2 myocardial cells in Control group and HG+PA group was performed,and the transcri ptome sequencing results were analyzed.3.Bodypy immunofluorescence detection of lipid levels in H9c2 myocardial cells induced by high glucose and high fat.3.Western blot was u sed to detect the changes in Lc3 protein levels in H9c2 cardiomyocytes induced by high glu cose and high fat,and to observe the effects of autophagy inducer rapamycin(RAPA)and a utophagy inhibitor baflomycin(Baf A1)on lipid levels in H9c2 cardiomyocytes.4.Immuno fluorescence co localization experiment was used to observe the co localization of Lc3 and lipid droplets.5.Dot-blot method was used to detect changes in mRNA methylation level o f m6 A.qPCR was used to screen for changes in m6 A methylase Wtap mRNA,Mettl3 mRN A,Mettl14 mRNA,and Alkbh5 mRNA,and Western blot was used to detect related protein levels.6.Bodipy immunofluorescence observation of changes in lipid levels in H9c2 myoc ardial cells induced by high glucose and high fat after overexpression of Alkbh5.7.Change s in lipid levels after overexpression of Alkbh5 and si Alkbh5.8.The binding ability of Alkb h5 and Atg7 mRNA and the change of m6 A level in Atg7 mRNA were detected by RIP exp eriment,and the level of Atg7 protein was further determined by Western blot.8.Predict th e m6 A site in Atg7 mRNA through the website and verify it through the fluorescent plum re porter gene.Animal experiment: Forty C57BL/6 male mice(20 ± 2g)aged 8 weeks were randomly divided into Control group,DCM group,VEC group,and Alkbh5 group,with 10 mice in ea ch group.Control group and VEC group were fed with regular feed,DCM group and Alkbh5 group were fed with 60% high fat feed,and 20 mice in DCM group and Alkbh5 group we re injected with STZ intraperitoneally at a dose of 60mg/kg for 5 consecutive days.In the 11 th week,pAAV-cTNT-Bd Breen WPRE and pAAV-cTNT-Alkbh5-3x FLAG-P2A-Gd Green WPRE viruses were injected into VEC and Alkbh5 mice through the tail vein of mice,respe ctively.The pAAV-cTNT-Bd Breen WPRE virus titers were 1.38e+13,and the pAAV-cTNTAlkbh5-3x FLAG-P2A-Gd Green WPRE virus titers were 1.67e+13.Each mouse was injecte d with 50 ul.After 16 weeks,the mouse heart tissue was extracted after ultrasound measure ment of cardiac function.Ultrasound was used to detect cardiac function indicators such as LVESD,LVEDD,LVEF,and LVFS in mice,HE staining was used to detect pathological ch anges in left ventricular myocardium,Masson staining was used to detect left ventricular m yocardial fibrosis,qPCR was used to detect changes in ANP mRNA and BNP mRNA levels in mouse heart tissue,ELISA was used to detect changes in LDH,CK-MB,cTN1 in mouse serum,and the effect of expressed Alkbh5 on Atg7 and Lc3 protein levels was verified at t he animal level.Part 3:Cell experiment: 1.cck8 screen the best working concentration of Shengmaiyin on H9c2 myocardial cytoprotection induced by high glucose and high fat.2.qPCR detection of the levels of ANP mRNA and BNP mRNA in H9c2 myocardial cells induced by high glucose a nd high fat with Shengmai Yin.3.Western blot was used to detect the expression levels of Alkbh5,Atg7,and Lc3 proteins in H9c2 myocardial cells in each group.4.Bodypy staining detection of lipid droplet content and co localization of lipid droplets with Lc3 in H9c2 my ocardial cells in each groupAnimal experiment: 1.Western blot detection of the expression levels of Alkbh5,Atg7,and Lc3 proteins in myocardial cells of mice in each group.2.Bodypy staining was used to detect the lipid droplet content in myocardial cells of mice in each group.ResultPart 1:1.The results of cardiac ultrasound examination showed that the left ventricular end sy stolic diameter(LVESD)and left ventricular end diastolic diameter(LVEDD)of DCM mic e significantly increased,while the left ventricular ejection fraction(LVEF)and short axis s hortening rate(LVFS)significantly decreased,indicating cardiac systolic dysfunction in D CM mice.Shengmai Yin can significantly reduce LVESD and LVEDD,increase LVEF and LVFS,effectively improve DCM induced cardiac dysfunction in mice,and reduce ventricul ar remodeling.2.Compared with the Control group,the DCM group showed disordered arrangement o f myocardial cells,hypertrophy of myocardial cells,disappearance of striations,and myocar dial interstitial edema.Compared with the DCM group,the HSMY and LSMY groups show ed intact myocardial tissue,slight swelling of myocardial cells,and slightly blurred striation s.Among them,the high-dose group had the least pathological changes in myocardial tissu e,which was close to the control group.Shengmai Yin could significantly improve the path ological disorder of mouse heart tissue.3.Serum indicators LDH,CK-MB,and cTN1 were significantly increased in DCM gro up mice,and different doses of Shengmai Yin were able to inhibit the increase of these indi cators,and high-dose Shengmai Yin had a more significant effect.4.Elevated levels of ANP mRNA and BNP mRNA were detected in both the hearts of DCM mice and H9c2 cardiomyocytes induced by high glucose and high fat.The interventio n of Shengmai Yin can effectively restore the levels of ANP mRNA and BNP mRNA.Part 2:1.In Bodypy immunofluorescence staining results,high glucose and high fat induced li pid accumulation in H9c2 cardiomyocytes.Western blot detection of Lc3-II protein levels r evealed a decrease in autophagy levels.In immunofluorescence staining,Lc3 and lipid drop lets were found to be co localized,confirming the occurrence of lipid autophagy in H9c2 ca rdiomyocytes.2.Through Dot blot experiments,it was found that high sugar and high fat induced a si gnificant increase in m6 A methylation levels in H9c2 myocardial cells.qPCR and Western blot screening showed that high sugar and high fat induced changes in m6 A methylation en zymes in H9c2 myocardial cells,and it was found that Alkbh5 demethylase was significantl y reduced.3.Compared with the HG+PA group,overexpression of Alkbh5 resulted in a decrease in lipid levels in Bodypy immunofluorescence staining.After si Alkbh5 transfection,Bodypy i mmunofluorescence staining significantly increased lipid levels compared to the Control gr oup.4.In RIP experiment,it was found that Alkbh5 protein could target Atg7 mRNA,and in H9c2 cardiomyocytes induced by high glucose and high fat,the binding ability of Alkbh5 t o Atg7 decreased,and the level of m6 A methylation in Atg7 mRNA increased.5.After overexpression of Alkbh5,it was found that the levels of Lc3 and Atg7 proteins increased in mouse myocardial tissue and H9c2 cells.6.Overexpression of Alkbh5 in animal mice can significantly reduce LVESD and LVE DD,increase LVEF and LVFS,significantly reduce CVF,significantly decrease ANP mRN A and BNP mRNA levels in myocardial tissue,and significantly decrease LDH,CK-MB,a nd cTN1 levels in serum.Part 3:1.The best working concentration of Shengmaiyin(CCk8)to screen the protective effe ct of Shengmaiyin on H9c2 myocardial cytoprotection induced by high glucose and high fat is 10ul/ml2.Western blot was used to detect the expression of Alkbh5,Lc3,and Atg7 proteins in H9c2 cells and animal myocardial tissue.It was found that Maiyin significantly increased t he levels of Alkbh5,Lc3,and Atg7 proteins.3.In the Bodypy immunofluorescence experiment of animal heart tissue and cells,Shen gmai Yin inhibits lipid accumulation.Conclusion1.The decrease in lipid autophagy levels is the main cause of lipid accumulation in the heart of DCM mice,and promoting lipid autophagy can effectively improve myocardial da mage in DCM mice.2.Alkbh5 is the main m6 A methylase in the process of DCM,which can target Atg7 mRNA and regulate lipid autophagy levels by modifying the 2164 site in the CDS region and the 30 site in the 5’UTR region of Atg7 mRNA for demethylation.3.The protective effect of Shengmai Yin on the heart of DCM is to promote the occurre nce of lipid autophagy through the Alkbh5-Atg7 pathway,thereby effectively alleviating m yocardial damage in DCM.